Simultaneous Analysis of Catecholamines and Metanephrines in Urine by LC-MS/MS

By Shun-Hsin Liang and Frances Carroll

Abstract

Clinical diagnosis of pheochromocytoma and paraganglioma is often based on the analysis of catecholamines (epinephrine, norepinephrine, dopamine) and metanephrines (metanephrine, normetanephrine, 3-methoxytyramine) in urine. Analysis of these polar compounds using reversed-phase LC can be difficult due to limited chromatographic retention, which results in poor separation of the analytes from closely eluting matrix interferences. This method overcomes these problems by combining a simple solid phase extraction procedure with the consistent and accurate chromatographic performance of a Raptor Biphenyl column.

Introduction

Pheochromocytomas and paragangliomas are neuroendocrine tumors that are characterized by the release of elevated levels of catecholamines. The Endocrine Society, the American Association for Clinical Chemistry, and the European Society for Endocrinology have all released clinical practice guidelines for the diagnosis and management of these diseases and recommend two major diagnostic tests: (1) plasma free metanephrines, which is highly sensitive, and (2) 24-hour urinary collection of catecholamines and metanephrines, which is highly specific.

The target analytes for urinary analysis are epinephrine (EPI), norepinephrine (NE), and dopamine (DA), as well as their respective methylated metabolites, metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) (Figure 1). Although reversed-phase LC-MS/MS has been the method of choice for this analysis, challenges still remain because these polar analytes are difficult to retain and this, in combination with the presence of matrix interferences, can result in inconsistent chromatographic performance. To solve these problems, a simple and fast solid phase extraction (SPE) procedure was developed, followed by LC-MS/MS analysis using a Raptor Biphenyl column. The method is accurate and robust for the simultaneous analysis of catecholamines and metanephrines in urine and, as such, is suitable for high-throughput clinical diagnostics.

Figure 1: Structures of Catecholamines and Methylated Metabolites

Experimental

Solid Phase Extraction
A 200 µL aliquot of urine sample was mixed with 10 μL of internal standard solution (1 µg/mL in methanol) and 600 μL of 250 mM ammonium acetate solution. The mixture was loaded onto the EVOLUTE EXPRESS WCX 96-well plate (30 mg) and washed with 1 mL water and 1 mL methanol:acetonitrile (60:40). The elution was performed with 200 µL of water:methanol (95:5) solution containing 5% formic acid and then 2 μL was injected for analysis.

Calibration Standards and Quality Control Samples
DC Mass Spect Gold Urine (Golden West Biologicals) was fortified with six analytes to prepare calibrated standards and QC samples. The linearity ranges were from 0.5-250 ng/mL for epinephrine; 1-1,000 ng/mL for norepinephrine; 5-1,500 ng/mL for metanephrine and 3-methoxytyramine; and 10-2,000 ng/mL for dopamine and normetanephrine. Three QC levels were prepared at 2.5, 25, and 75 ng/mL for epinephrine and norepinephrine; 25, 75, and 750 ng/mL for metanephrine and 3-methoxytyramine; and 75, 750, and 1,500 ng/mL for dopamine and normetanephrine. The fortified standard and QC samples were subjected to the SPE procedure described above.

Urine Analysis
To confirm that the validated method could accurately measure the urinary catecholamines and metabolites, two levels of urine samples (Bio-Rad Lyphochek quantitative urine controls) were analyzed with the established SPE and chromatographic methods. The normal urine was fortified with 10 ng/mL of epinephrine, norepinephrine, 3-methoxytyramine, and 100 ng/mL of dopamine, metanephrine, and normetanephrine. The abnormal urine was fortified with 50 ng/mL of epinephrine and norepinephrine and 200 ng/mL of dopamine, metanephrine, normetanephrine, 3-methoxytyramine. Accuracy was determined using the measured concentration difference between the blank and fortified urine.

LC-MS/MS analysis of catecholamines and metanephrines in urine was performed on an ACQUITY UPLC instrument coupled with a Waters Xevo TQ-S mass spectrometer. Instrument conditions were as follows and analyte transitions are provided in Table I.

Results and Discussion

Chromatographic Performance
The analysis of normal human urine (Bio-Rad Lyphochek quantitative urine control, level 1) demonstrates that a fast 5-minute chromatographic analysis is achieved with direct injection of the elution solution that was obtained from the simple SPE procedure (Figure 2). The Raptor Biphenyl column provided adequate retention such that all target analytes could be quantified with no observed influence from matrix interferences.

Linearity
Good linearity was achieved for all compounds using a 1/x weighted linear regression for epinephrine, norepinephrine, dopamine, metanephrine, and normetanephrine, and a 1/x quadratic regression for 3-methoxytyramine (Figure 3). All six analytes had r2 values of 0.999 or greater and deviations of <10%, except for the lowest concentration standard, which had deviations of <20%.

Figure 3: Calibration Curves

Accuracy and Precision
Precision and accuracy analyses were performed on three different days. The accuracy of the method was demonstrated by the recovery values, which were within 10% of the nominal concentration for all QC levels. The %RSD was 0.2-6.2% and 1.8-3.8% for intraday and interday comparisons, respectively, indicating that method precision was also acceptable (Table II).

Table II: Accuracy and Precision of QC Samples

QC Level 1 (2.5–75 ng/mL)

QC Level 2 (25–750 ng/mL)

QC Level 3 (75–1,500 ng/mL)

Analyte

Average Conc. (ng/mL)

Average Accuracy (%)

%RSD

Average Conc. (ng/mL)

Average Accuracy (%)

%RSD

Average Conc. (ng/mL)

Average Accuracy (%)

%RSD

Epinephrine

2.43

97.3

2.97

26.0

104

2.31

77.9

104

2.19

Norepinephrine

2.44

97.5

2.34

25.3

101

2.92

76.9

103

2.48

Dopamine

74.5

99.4

3.42

787

105

3.85

1,560

104

3.37

Metanephrine

25.4

102

2.46

81.0

108

1.81

774

103

2.00

Normetanephrine

78.4

105

3.36

788

105

2.98

1,570

105

3.52

3-Methoxytyramine

25.7

103

3.39

81.4

109

2.87

764

102

2.94

Urine Analysis
From three sets of analyses of blank and fortified urine samples (BioRad Lyphochek quantitative urine control, level 1 and level 2), the results showed that the recoveries of all fortified samples were within 90-105% of their nominal values (Table III). This demonstrated that the validated method is suitable for the analysis of catecholamines and metanephrines in urine from human patients at clinically relevant levels.

Table III: Analysis of Fortified Urine Samples

Level 1

Level 2

Analyte

Blank Urine (ng/mL)

Fortified Urine (ng/mL)

Calculated Conc. (ng/mL)

Fortified Conc. (ng/mL)

Average Accuracy (%)

Blank Urine (ng/mL)

Fortified Urine (ng/mL)

Calculated Conc. (ng/mL)

Fortified Conc. (ng/mL)

Average Accuracy (%)

Epinephrine

13.0

22.6

9.57

10.0

95.7

91.7

143

51.4

50.0

103

Norepinephrine

42.2

51.3

9.14

10.0

91.4

185

234

49.3

50.0

98.7

Dopamine

77.2

166

90.2

100

90.2

459

655

196

200

98.1

Metanephrine

71.9

163

90.7

100

90.7

516

699

183

200

91.7

Normetanephrine

251

345

94.8

100

94.8

1,310

1,500

188

200

94.3

3-Methoxytyramine

12.2

21.5

9.31

10.0

93.1

418

603

185

200

92.6

Conclusion

As demonstrated here, the Raptor Biphenyl column provides good retention and accurate results for the simultaneous analysis of catecholamines and metanephrines in urine. With a fast, simple sample preparation procedure and just five minutes of chromatographic analysis time, the established method is recommended for high-throughput labs supporting the clinical diagnosis of pheochromocytoma and paraganglioma.